Analysis of biological samples often involves methods in which a color is developed that is proportional to the amount of an analyte in the sample. For example, enzymes may be employed to oxidize the analyte of interest and the extent of reaction is shown by a color change of an indicator compound. Of particular interest herein is the family of tetrazolium salts that is used as indicators. Such salts are reduced to formazan dyes by reducing substances. To measure an analyte, an enzyme (e.g., an NAD-dependent dehydrogenase enzyme), oxidizes the analyte to produce the reduced form (e.g., NADH), that reacts with a tetrazolium salt to produce a colored formazan. A mediator may be needed to facilitate the reaction. Since the amount of NADH produced by the analyte reaction is proportional to the amount of formazan produced, the amount of the analyte can be measured indirectly by the color formed.
Tetrazolium salts have been used in various applications. But, in particular, they have been used in the medical field for measuring the analytes in various biological fluids such as blood, urine, plasma, and serum. These indicators often are used with reagent systems placed on test strips which, when in contact with a fluid sample, react with the analyte of interest and display a color that indicates the amount of the analyte present. Although in some instances, the color change can be read visually by reference to color charts, more accurate readings may be made spectrophotometrically by instruments that are designed for that purpose. Typically, light is directed onto the test strip and the reflected light is measured to determine the effect of the color change on the strip.
Tetrazolium salts should produce formazans that absorb light at wave lengths that avoid interference by substances in the sample, such as the hemoglobin in whole blood. Consequently, a family of thiazolyl tetrazolium salts has been developed that produce formazans that absorb light having wavelengths above about 640 nm, such as those produced by LEDs used as light sources. LEDs provide a narrow range of wavelengths that vary only about ±5 nm. Such thiazolyl tetrazolium salts are disclosed in several U.S. patents such as U.S. Pat. Nos. 5,126,275; 5,322,680; 5,300,637; and 5,290,536.
Most biological samples are aqueous in nature, so it is desirable that the indicators be soluble in the sample. However, many of the tetrazolium salts are not very soluble. One supplier of tetrazolium salts, Dojindo Laboratories, has a line of indicators that have been made more soluble by the addition of sulfonic acid groups to the indicator molecule. See U.S. Pat. No. 6,063,587 and published Japanese patent applications JP58113181 A2 and JP58113182 A2. Their WST series of tetrazolium salt indicators are often mentioned in patents disclosing analytical methods. One example is found in U.S. Pat. No. 6,586,199. The solubility of the WST series of tetrazolium salts is reported to be about 10 mg/mL water. Solubility also may be increased when certain sulfonate and phosphonate counterions of tetrazolium salts are used such as disclosed in U.S. Pat. No. 5,250,695.
Other patents discussing tetrazolium salt indicators include EP0476-455 B1; US 2004/0132004 A1; WO 98/37157; U.S. Pat. Nos. 6,183,878 B1; 6,207,292 B1; 6,277,307 B1; DE 21 47 466; U.S. Pat. Nos. 5,185,450; 5,196,314.
The present inventors wanted to improve the solubility of the thiazolyl tetrazolium salts while retaining their ability to provide a relatively flat spectral response from their formazans in the region of 600 to 640 nm in response to incident light provided by LEDs. As will be seen in the description of the invention below, they have succeeded in providing thiazolyl tetrazolium salts having greater solubility, while retaining the desired spectral response.